Chagas disease, caused by Trypanosoma cruzi, is globally ranked behind malaria and schistosomiasis as the third most serious parasitic disease, and remains the most serious parasitic disease in Latin America in terms of disability adjusted life years. Despite recent progress controlling dissemination of T. cruzi in reduviid bugs, no treatment is available for chronic Chagas disease, and acute infections can only be treated with highly toxic drugs. Thus, there is a critical need for new approaches to treatment of T. cruzi infections. Metacyclogenesis is the process by which non-infectious "insect form" epimastigotes of T. cruzi differentiate into infectious metacyclic trypomastigotes. This process is the subject of much interest among scientists because of the implications for treatment/prevention of Chagas disease. We have used an axenic model system for metacyclogenesis, begun a nonredundant microarray of sequence-verified cDNA sequences, and established an exogenously regulated expression system that can be used to dissect this important differentiation system. The specific aims of the project are: 1) To establish and validate comprehensive microarrays of T. cruzi genes expressed during differentiation of epimastigotes into metacyclic trypomastigotes. 2) To use these DNA microarrays to identify T. cruzi genes that are differentially expressed during metacyclogenesis. 3) To use known potentiators of T. cruzi metacyclogenesis to identify 5-10 candidate genes that trigger, control or direct the differentiation process. 4) To document the function of these regulatory genes by in silico analysis, overexpression, knockout of the endogenous genes, or regulated inhibition of expression (e.g., using RNAi), using our regulated T. cruzi gene expression system. We anticipate identifying several hundred differentially regulated genes in Specific Aim #2. In Specific Aim #3, we will screen out most of the genes that are not directly causal in the differentiation process and select 5-10 candidates for regulatory genes for further analysis. In Specific Aim #4, we will use our regulatable T. cruzi genetic system and RNAi to establish the functions and roles of the differentially expressed genes that play a probable